Oxygen is one of the most abundant elements and is present in almost all biologically relevant molecules. In the past the NMR active isotope 17O has received little attention, due to short relaxation times and low sensitivity. However, with the availability of higher magnetic field strengths, the use of 17O-enriched substrates and the favorable T2 */T1 ratio, any sensitivity concerns are quickly overcome, allowing the acquisition of 3D MRSI data in the span of seconds. Furthermore, the wide chemical shift dispersion allows the detection of a wide range of metabolites. Here we propose to develop 17O NMR in combination with 17O-enriched substrate infusion into a fast, sensitive and robust method to spatially map metabolic fluxes in the rat brain in vivo. Metabolism of 17O-glucose enriched in the 1, 2 or 3 positions gives information on glycolytic, tricarboxylic acid cycle or pyruvate dehydrogenase activity, respectively. Astroglial metabolism and pyruvate carboxylase activity can be assessed with 17O-enriched acetate and bicarbonate. The 17O turnover characteristics of each substrate can be described by a metabolic model, some of which require additional inputs such as recirculated 17O-labeled water from other organs. Here we will develop the methods to measure all required inputs in order to reliably obtain the desired metabolic fluxes. Following the optimization of 17O substrate synthesis, the characteristics of 17O NMR and 17O label dynamics will be studied in vitro. The established 1H-[13C]-NMR technique in combination with 13C-labeled glucose and 2-deoxyglucose infusions will be used to validate the 17O-based cerebral metabolic fluxes through the tricarboxylic acid cycle and glycolysis, respectively, in the rat brain in vivo at 11.7 T.